Cerebral Cortex April 2005;15:448--459 doi:10.1093/cercor/bhh147 Advance Access publication August 5, 2004 Localization of the GLYT1 Glycine Beatriz Cubelos, Cecilio Gime´nez and Francisco Zafra Transporter at Glutamatergic Synapses Centro de Biologı´a Molecular ‘Severo Ochoa’, Facultad de in the Rat Brain Ciencias, Universidad Auto´noma de Madrid, Consejo Superior de Investigaciones Cientı´ficas, Madrid, Spain In this study, we present evidence that a glycine transporter, GLYT1, responses to NMDA both in vitro and in vivo (Chen et al., 2003; Downloaded from https://academic.oup.com/cercor/article/15/4/448/351216 by guest on 24 September 2021 is expressed in neurons and that it is associated with glutamatergic Kinney et al., 2003). Recent evidence obtained in knockout mice synapses. Despite the presence of GLYT1 mRNA in both glial cells and for glycine transporter genes confirmed the involvement of both in glutamatergic neurons, previous studies have mainly localized GLYT1 and GLYT2 in glycinergic inhibitory neurotransmission. GLYT1 immunoreactivity to glial cells in the caudal regions of the In these mutant mice, glycinergic neurotransmission was largely nervous system. However, using novel sequence specific antibodies, altered, leading to the suggestion that GLYT1 might remove we have identified GLYT1 not only in glia, but also in neurons. The glycine from the synaptic cleft, whereas GLYT2 would replenish immunostaining of neuronal elements could best be appreciated in the presynaptic pool of glycine (Gomeza et al., 2003a,b). The forebrain areas such as the neocortex or the hippocampus, and it was possible role of GLYT1 in glutamatergic neurotransmission in found in fibers, terminal boutons and in some dendrites. Double these GLYT1 deficient mice was not addressed since they died labeling confocal microscopy with the glutamatergic marker vGLUT1 shortly after birth. However, heterozygous animals that ex- revealed an enrichment of GLYT1 in a subpopulation of glutamatergic pressed only 50% of GLYT1 had an enhanced hippocampal terminals. Moreover, through electron microscopy, we observed an NMDA receptor function and memory retention. These animals enrichment of GLYT1 in both the presynaptic and the postsynaptic were also protected against an amphetamine disruption of aspects of putative glutamatergic terminals that established asym- sensory gating, suggesting that drugs which inhibit GLYT1 metric synapses. In addition, we demonstrated that GLYT1 was might have both cognitive enhancing and antipsychotic effects physically associated with the NMDA receptor in a biochemical (Tsai et al., 2004). assay. In conclusion, the close spatial association of GLYT1 and In order to fully clarify the role of GLYT1 in glutamatergic glutamatergic synapses strongly supports a role for this protein in neurotransmission, it is necessary to determine the precise neurotransmission mediated by NMDA receptors in the forebrain, and cellular and subcellular localization of this transporter. Previous perhaps in other regions of the CNS. histochemical studies revealed a discrepancy between the distribution of GLYT1 mRNA and protein. While GLYT1 mRNA Keywords: glycine, glutamate, immunohistochemistry, NMDA receptor transcripts were detected in glia and many types of neuron throughout the nervous system, the protein was only detected in glial cells (Zafra et al., 1995a,b). Introduction Here, we present detailed immunohistochemical data show- Beside its well-characterized role as an inhibitory neurotrans- ing that, in addition to the previously described expression of mitter, glycine is a co-agonist of NMDA receptors that is GLYT1 in glia, this transporter is also expressed at the pre- and necessary both for ion channel opening and probably for the postsynaptic aspects of glutamatergic synapses. This association internalization of the receptor from the cell surface (Kemp and was especially evident in the neocortex and hippocampus, areas Leeson, 1993; Nong et al., 2003). While it was initially believed where GLYT1 formed immunoprecipitable complexes with that the concentration of glycine in the synaptic cleft would be NMDA receptors. These results indicate a close relationship high enough to saturate the glycine site on the NMDA receptor, between GLYT1 and glutamatergic neurotransmission. recent pharmacological and electrophysiological evidence in- dicates that this might be not the case. Indeed, the levels of Materials and Methods glycine in the synaptic cleft could be close to the threshold levels for activation of the receptor (Supplisson and Bergman, Materials 1997; Berger et al., 1998; Bergeron et al., 1998; Kew et al., 1998, Oligonucleotides were synthesized by Isogen (Utrecht, NL). Bovine 2000). The concentration of glycine in the synaptic cleft is serum albumin (BSA), ethylene glycol-bis-aminoethyl ether N,N,N9,N9- regulated by the glycine transporters GLYT1 and GLYT2 (for tetraacetic acid, N-2-hydroxyethylpiperazine-N9-2-ethane sulfonic acid (HEPES), ovalbumin, diaminobenzidine, phenylmethanesulfonyl fluoride a review see Lo´pez-Corcuera et al., 2001). GLYT2 is predom- (PMSF), Triton X-100, Trizma base, Freund’s adjuvant and glutathione inantly found in the spinal cord and the brainstem, associated were from Sigma (St Louis, MO). Dextran-70, pGEX2, Glutathione with glycinergic neurotransmission (Luque et al., 1995; Zafra Sepharose 4B, standard proteins for sodium dodecyl sulfate--polyacryl- et al., 1995a,b; Jursky and Nelson, 1996). However, while GLYT1 amide gel electrophoresis (SDS--PAGE) (Rainbow markers), biotinylated is also expressed highly in glycinergic areas, GLYT1 mRNA was donkey anti-rabbit immunoglobulin Gs (IgGs), streptavidin-biotinylated also found in areas devoid of inhibitory glycinergic neuro- horseradish peroxidase and the enhanced chemioluminescent detec- tion method (ECL) were from Amersham-Pharmacia (Amersham, UK). transmission where it might play a role in NMDA-mediated Isopropylthiogalactoside, EcoRI, BamHI and T4 ligase were from Roche neurotransmission (Smith et al., 1992). In support of this (Germany). Lipofectamine-PLUS was from Invitrogen and Taq poly- hypothesis, N[3-(49-fluorophenyl)-3-(49-phenylphenoxy)propyl]- merase from Perkin Elmer Cetus. Affi-Gel 15 (N-hydroxysuccinimide sarcosine (NFPS), a specific inhibitor of GLYT1, potentiates the esters of crosslinked agarose) and nitrocellulose sheets were from Cerebral Cortex V 15 N 4 Ó Oxford University Press 2004; all rights reserved BioRad (Richmond, CA). Newborn calf serum was from Gibco (Paisley, Electrophoresis and Blotting UK). Guinea pig anti-vesicular glutamate transporters 1 and 2 (anti- SDS--PAGE was done in the presence of 2-mercaptoethanol. The gels vGLUT1 and anti-vGLUT2) were from Chemicon (Temecula, CA) and were run slowly (overnight) at constant current starting at 30 V. After mouse anti-NMDA from Phamingen. Gold-coupled antibodies were from electrophoresis, samples were transferred by electroblotting onto Auroprobes. Goat anti-rabbit, goat anti-guinea pig and goat anti-mouse a nitrocellulose membrane in a semidry electroblotting system (LKB) coupled to Alexa Fluor 488 or Alexa Fluor 594 were from Molecular at 1.2 mA/cm2 for 2 h. The transfer buffer consisted of 192 mM glycine Probes (Eugene, OR). TAAB 812 was from TAAB laboratories (Reading, and 25 mM Tris--HCl, pH 8.3. Nonspecific protein binding to the blot was UK) and Lowicryl HM20 from Agar Scientific Ltd (Stanstead, UK). All blocked by the incubation of the filter with 5% non-fat milk protein in other reagents were analytical grade. 10 mM Tris--HCl, pH 7.5, 150 mM NaCl for 4 h at 25°C. The blot was then probed with the indicated dilutions of crude antisera or purified Methods antibodies overnight at 4°C. After washing, blots were then probed with an anti-rabbit IgG peroxidase linked, and bands were visualized Preparation of Glutathione S-transferase--Glycine Transporter with ECL and quantified by densitometry (Molecular Dynamics Image Fusion Proteins Quant v. 3.0). The 111 bp cDNA fragment encoding the amino terminus of GLYT1b was synthesized by polymerase chain reaction using the full-length Downloaded from https://academic.oup.com/cercor/article/15/4/448/351216 by guest on 24 September 2021 clone as template. According to available experimental evidence, the Immunocytochemistry N-terminal domain of GLYT1 is intracellularly placed (Olivares et al., Five adult rats (3 months old) (Wistar strain) were deeply anesthetized 1997). The specific oligonucleotide primers were CGGGATC- with pentobarbital (100 mg/kg) and perfused through the left ventricle-- CATGGCTGTGGCTCACGGA and GGAATTCACTCGATCTGGTTGCCC- aorta with a fixative solution containing formaldehyde (4 or 0.5%) in CA. Each primer consisted of 18 nucleotides of the GLYT1b sequence 0.1 M sodium phosphate pH 7.4 (500 ml, room temperature), preceded plus the restriction sites either for BamHI or EcoRI in their 59 ends. The by a brief flush (10--15 s) of Dextran-70 (MW 70000; 20 mg/ml) in the BamHI site was placed such that the inserts were in the correct reading same buffer without fixative. The right atrium was cut open at the start frame after insertion in BamHI/EcoRI sites of the pGEX-2 plasmid. These of the perfusion. The perfusion liquids were delivered by a peristaltic restriction sites are located in the carboxyl terminus of the glutathione S- pump at 50 ml/min, starting within 20 s after thoracotomy. The brains transferase (GST). After ligation of the inserts to pGEX-2, Escherichia were postfixed (overnight) in the same fixative. The tissue was stored at coli